In unfolded proteins, peptide bonds involving Pro residues exist in an equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate limiting step in protein folding studies. Moreover, kinases and phosphatases that act upon Ser/Thr-Pro motifs exhibit preferential recognition of either the cis- or the trans-Pro state. Using NMR spectroscopy at both atmospheric and high pressure, population of cis-Pro falls well below previous estimates, an effect we attributed to the use of short peptides with charged termini in most prior model studies. For the intrinsically disordered protein -synuclein, cis-Pro populations at all of its five X-Pro bonds were found to be less than 5%, with only modest ionic strength dependence and no detectable effect of the previously demonstrated interaction between the N- and C-terminal halves of the protein. Comparison to small peptides with the same amino acid sequence indicated that peptides, particularly those with unblocked, oppositely charged amino and carboxyl end groups, strongly overestimate the amount of cis-Pro. Our finding has important implications for the folding barriers and kinetic rates of folding in proteins that contain multiple Pro residues. The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. We studied the redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved a-crystallin domain, and provided the first structural characterization of a small heat-shock protein monomer. While HSP27 assembles into oligomers, we showed that the transiently populated monomers released upon reduction are highly active chaperones in vitro, but are kinetically unstable and susceptible to uncontrolled aggregation. By using relaxation dispersion and high pressure NMR experiments, we revealed that the pair of beta-strands that mediate dimerization become partially disordered in the monomer and enhance its chaperone activity. Strikingly, we found that numerous HSP27 mutations associated with inherited neuropathies cluster to the unstructured region in the monomer. Among mammalian small heat shock proteins, the high degree of sequence conservation in the alpha-crystallin domain suggests that partial unfolding of the monomeric state may be a general feature of this class of chaperones. Although the a-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a proteins structure. Highly charged single a-helical (SAH) domains, consisting of a high percentage (>75%) Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. We studied the 68-residue medial tail domain of myosin-VI and found it to contain a highly ordered a-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15-150), small (ca 4 Hz) 3JHNHa couplings, and a near-perfect fit to an ideal model a-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous than the protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. 15N relaxation data indicate a very high degree of rotational diffusion anisotropy (Dparr / Dperp 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight a-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region showed a decrease in alignment as the distance from the tagging site increased. This decrease yielded a precise measure for the persistence length of 22410 at 20 C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin VI lever arm.